Project Summary Our long-range goals are to discover novel therapeutic strategies that advance treatment of cancer by targeting the ubiquitin-proteasome system, which is the major cellular pathway for selective degradation of proteins. Recently, we developed a new high-throughput screening assay to identify small molecule antagonists or agonists that target the catalytic activity of the Cullin-RING E3 ubiquitin ligases (CRL), which direct numerous protein substrates for degradation by the 26S proteasome. Our new technology is based on fluorescence (Frster) resonance energy transfer (FRET). It detects FRET signals that are produced by ligation of two fluorescently labeled ubiquitin (Ub) molecules, a reaction driven by CRL's core ligase complex. This assay has already been adapted for use on a robotic system to screen a large collection of small molecules, resulting in identification of a group of inhibitors with cytotoxic activity against tumor cells. Using the established methods, this project will carry out additional screens and identify a large set of small molecule modulators. We will subject these hits to a variety of biochemical, biophysical and cell-based assays to remove false positives and to classify them into distinct classes of inhibitors. Cheminformatics, structural biology, and medicinal chemical approaches will follow to refine such small molecules to increase their targeting potency. It is hopeful that this project will lead to discovery of small molecules with `drug-like' properties capable of selectively killing cancer cells.